The free radical chain oxidation of polyunsaturated fatty acids (PUFAs) and their biologically relevant esters is a complex reaction giving scores of possible products from a single molecular species. This process, known as lipid peroxidation, is a hallmark of diverse environmental chemical exposures, drug toxicities and stresses. In addition to the many peroxide products that form from polyunsaturated lipids, a set of very reactive electrophiles is also generated, 4-hydroxy-2-nonenal (HNE) being the most studied example. These electrophilic residuals of lipid peroxidation modify nucleic acids and proteins and in this way,the consequences of lipid oxidative degradation is distributed to other important biomolecules. This proposal outlines experiments that probe the chemical mechanisms of lipid peroxidation, develops methods for the analysis of products formed in this process, provides a framework for understanding the oxidation of highly unsaturated lipids present in fish oils, and examines new phenolic antioxidants more potent than vitamin E. The proposed research is based on the hypothesis that the chemical mechanisms of lipid peroxidation and the formation of electrophilic byproducts that are a hallmark of this process can be rationally defined. Protocols developed to profile the important lipid peroxidation products formed from w-6 (linoleic and arachidonic) and w-3 (eicosapentenoic and docosahexenoic) fatty acids provide intimate insight into the process. Powerful HPLC/MS/MS methods permit the determination of individual products derived from particular molecular species even though the product is only a small part of a very complex mixture. Peroxidation profiling of human LDL and rodents exposed to an oxidative stress will be carried out and new powerful pyridinol antioxidants will be studied in "proof of concept" in vivo experiments with rodents. The same HPLC/MS/MS methods used for peroxidation profiling can provide profiles of electrophiles derived from the peroxide primary products. The spectrum of products formed from highly unsaturated w-3 fatty acids and the mechanism for their formation will be investigated. The electrophiles identified from phospholipids will form the basis of a screening program in collaboration with Projects 3 and 4 of the Program Project.